Amphotericin B liposome preparation

ABSTRACT

A novel composition and method for solubilizing amphiphilic drugs in a small amount of organic solvent for use in improved liposomes is disclosed. A phosphatidylglycerol is acidified in a small amount of organic solvent. The amphiphilic drug, such as Amphotericin B, suspended in organic solvent is then added to the acidified phosphatidylycerol and a soluble complex is formed between the phosphatidylglycerol and the amphiphilic drug. When the liposome composition incorporating the soluble complex is hydrated, the final pH of the hydrating aqueous buffer is carefully controlled. The Amphotericin B liposomes formed have markedly reduced toxicity.

This is a continuation of copending application Ser. No. 07/600/154filed Oct. 19, 1990 which is a continuation of Ser. No. 119,518 filedNov. 12, 1987, now abandoned.

FIELD OF THE INVENTION

This invention relates to a novel procedure for solubilizing amphiphilicdrugs. In another aspect, this invention relates to improved methods ofAmphotericin B liposome preparation. In another aspect this inventionrelates to improved methods of producing liposomes by a commerciallyfeasible process. This invention also relates to liposomal AmphotericinB having reduced toxicity and to a new method of treatment withliposomal Amphotericin B.

BACKGROUND OF THE INVENTION

Systemic fungal infections are a major cause of mortality in cancerpatients and other immunocompromised individuals. Unfortunately, fungalinfections very often defy treatment because the few drugs that destroyfungi are extremely toxic to the host. Because of the drugs' toxicity,the lowest possible effective doses should be given. Unfortunately,because the drugs are diluted in the blood, and because large amounts ofthe drugs are degraded, or excreted or taken up by uninfected tissue,large doses actually are and must be given if the treatment is to beeffective.

The preferred treatment for systemic fungal infections is primarilylimited to two groups of drugs: the polyene antibiotics such asAmphotericin B and nystatin, and the imidazoles, such as ketaconazoleand miconazole. The polyene antifungal antibiotics readily bind tosterol components of host cells causing disruption of the membrane, cellpermeability and lysis. Amphotericin B has thus been associated withacute hemolytic crisis. Further, because it is particularly toxic tokidney tissue, it has been associated with irreversible renal damage andeven kidney failure, at therapeutic dosage levels. Medoff, G.,Kabayashi, G. (1980) Strategies in treatment of systemic fungalinfections. New England Journal of Medicine 302: 145-55; Cohen, J.(1982) Antifungal chemotherapy. Lancet ii: 532-37; Graybill, J. R.,Craven, P. C. (1983) Antifungal agents used in systemic mycosis:activity and therapeutic use. Drugs 25: 41-62.

It is a major goal of medical research to overcome the problemspresented by the need for compromise between dosages high enough tocontrol infection on the one hand, and unacceptable damage to healthytissues on the other. Recently it has been discovered that needed dosesof medicine can be delivered to diseased tissue while bypassing healthytissue using certain liposomal formulations. Additionally, it has beenrecognized that medication can be incorporated into liposomes,microscopic delivery vesicles made, in part, of phospholipids. See U.S.Pat. No. 4,663,167--Composition and Method for Treatment of DisseminatedFungal Infections in Mammals, incorporated here by reference, andpending Vestar Research Inc. application Ser. No. 899,064, entitled"Improved Treatment of Systemic Fungal Infections With PhospholipidParticles Encapsulating Polyene Antifungal Antibiotics", alsoincorporated here by reference, which discloses liposomal deliveryvesicles made, in part, from phospholipids.

Phospholipids form closed, fluid filled spheres when mixed with water.Phospholipid molecules are polar, having a hydrophilic ionizable head,and a hydrophobic tail consisting of long fatty acid chains. Thus, whensufficient phospholipid molecules are present with water, the tailsspontaneously herd together to exclude the water while the hydrophilicphosphate heads form bonds with the water.

The result is a bilayer in which the fatty acid tails point into thenewly formed membrane's interior and the polar heads point toward theaqueous medium. The polar heads at one surface of the membrane pointtoward the liposome's aqueous interior and those at the other surfacepoint toward the aqueous exterior environment. It is this chemicaltendency to form liquid filled spheres that allows the liposome to beloaded with medication. As the liposomes form, water soluble moleculeswill be incorporated into the aqueous interior, and lipophilic moleculeswill tend to be incorporated into the lipid bilayer. Liposomes may beeither multilamellar, like an onion with liquid separating many lipidbilayers, or unilamellar, with a single bilayer surrounding an entirelyliquid center.

In studies of mice, Amphotericin B incorporated into liposomes has beenshown to treat systemic fungal infections more effectively than whengiven as the free drug. Liposomes are not themselves toxic, and theyprotect their loads from being degraded or diluted. Thus, liposomes arethought to deliver concentrated doses of antifungal antibiotic at thediseased tissue without the toxicity that would otherwise be associatedwith freely circulating drug. Therefore, liposomal Amphotericin B drugdoses can exceed the maximum tolerated dose of free Amphotericin B.Mehta, R. (1982). Liposomal Amphotericin B is toxic to fungal cells butnot to mammalian cells. Biochimica et Biophysica Acta 770: 230-34.Liposomal encapsulated Amphotericin B has also been shown to be aneffective treatment for murine systemic fungal infections, includingCandidiasis, Cryptococcosis, and Histoplasmosis. Graybill, J. R. et al(1983) Treatment of murine cryptococcosis with liposomal associatedAmphotericin B. Journal of Infectious Diseases 145: 748-52; Taylor, R.L. et al (1982) Amphotericin B in liposomes: A novel therapy forhistoplasmosis. American Review of Respiratory Diseases 125: 610-611.

Liposomal Amphotericin B has also shown effectiveness in human patients,life saving when other treatments have failed, including freelycirculating Amphotericin B. Systemic fungal infections are seen mostcommonly in people whose immune systems are compromised by disease orimmunosuppressive drug therapy. As previously mentioned, theseinfections are a common cause of death to victims of acquired immunedeficiency syndrome and to cancer patients undergoing chemotherapy. Thecausative agents of these fungal infections are often endogenous fungithat would be rendered harmless but for the patient's impairedresistance. Lopez-Berestein, G. et al (1987) Treatment of hepatosplenicCandidiasis with liposomal Amphotericin B. Journal of Clinical Oncology5: 310-17.

Unfortunately, because of the chemical properties of the polyeneantifungal antibiotics, it has heretofore not been possible to produceliposomal Amphotericin B in commercial quantities. These antifungalagents are called polyenes because they contain three to sevenconjugated double bonds in the aliphatic chain making up a large lactonering. The double bonds are incorporated into one side of the ring of 26to 44 carbon atoms and along the opposite side of the ring 6 to 12hydroxyl groups are present. Additionally, these molecules containspecific carboxylic acid groups and amine groups. Amphotericin B andnystatin, for example, possess both an aminosugar and a carboxylic acidgroup. The polyene regions of the molecules are of course hydrophobicand lipophilic while the polyol and ionizable regions are hydrophilicand lipophobic. As such, these molecules are called amphiphilic.Additionally, because of the carboxylic group and the amine group,Amphotericin B can act as a Lowry-Br.o slashed.nsted acid or protondonor, or as a Lowry-Br.o slashed.nsted base or proton acceptor. Thecombination of these functionalities makes polyenes very poorly solublein water and most organic solvents. Bennett, J. E. (1974) Chemotherapyof systemic mycoses. New England J. Medicine 290: 320-23.

It has been the persistent problem of insolubility of the polyeneantifungal antibiotics in general and of Amphotericin B in particularthat has previously limited the prior art. Liposomal Amphotericin Bformation was heretofore limited to generally two methods, describedbelow, neither feasible for commercial scale production and neithershowing long-term stability or as great a reduction in toxicity as thepreparations herein described.

One prior method requires that the Amphotericin B be first dissolved inlarge volumes of volatile organic solvent such as methanol. To thatsolution would then be added the lipid mixture dissolved in a volatileorganic solvent such as methanol and/or chloroform. The solvents wouldthen have to be removed from the mixture to form a lipid-Amphotericin Bfilm. Removal of solvents could be accomplished by a variety of methodsbut usually by evaporation to dryness in a round bottom flask undervacuum or nitrogen. Taylor, R. L. (1980) Amphotericin B in liposomes: Anovel therapy for histoplasmosis. Am. Review Respiratory Disease 125:610-11; Graybill, J. R. et al (1982) Treatment of murine crytococcosisdiseases. J. Infectious Diseases 145: 748-52; Lopez-Berestein, G. (1983)Treatment and prophylaxis in disseminated infection due to Candidaalbicans in mice with liposome-encapsulated Amphotericin B. J.Infectious Diseases 147: 939-45; U.S. Pat. No. 4,663,167--Compositionand method for treatment of disseminated fungal infections in mammals.The prior art methods thus required removal of large volumes of organicsolvent, and preparation of a lipid-Amphotericin B film, an extra stepeliminated by the present invention. Moreover, the prior art methodswere thus practicable only in discrete batches and were not amenable tothe continuous flow process of the current invention. These are twodisadvantages which the industry has long attempted to overcome.

In another method of forming liposomal Amphotericin B, the lipid mixtureis dissolved in chloroform or another solvent and deposited and dried onthe sides of a round bottom flask or vesicle surface. A solution ofAmphotericin B dissolved in a small amount of dimethyl sulfoxide wouldthen be added to the previously deposited lipid film. The resultingpreparation would thereafter have to be dialyzed against a bufferedsaline or other solution to remove the dimethyl sulfoxide andnon-intercalated Amphotericin B. The procedure was extremely timeconsuming and expensive, and typically resulted in incorporation of only70% of the initial Amphotericin B. Tremblay, C. et al (1984) Efficacy ofliposome-intercalated Amphotericin B in treatment of systemicCandidiasis in mice. Antimicrobial Agents and Chemotherapy 26: 170-73.

Prior to the present invention, it was not possible to dissolveAmphotericin B in small quantities of volatile solvent, such thatscaled-up production could be commercially feasible. The presentinvention also enables the dissolved Amphotericin B--phospholipidliposomal solution to be spray dried, thus making commercial quantitiespractical by elimination of the elaborate and time consuming stepsdetailed above.

Accordingly, one object of the present invention is to provide animproved process for the solubilization of amphophilic drugs.

Another object of the present invention is to provide an improvedprocess for the encapsulation of polyene antifungal antibiotics intoliposomes.

More specifically, an object of the present invention is to provide animproved process for the formation of liposomal Amphotericin B.

Yet another object of the present invention is to provide a commerciallyfeasible process for the production of liposomal Amphotericin B.

Yet another object of the present invention is to provide a process forthe formation of liposomal Amphotericin B with reduced toxicity.

A further object of the present invention is to provide new methods oftreatment with Amphotericin B.

The manner in which these and other objects are realized by the presentinvention will be apparent from the summary and detailed description setforth below.

SUMMARY OF THE INVENTION

According to the present invention, the steps of either evaporation ofthe large volumes of volatile solvent into which the antibiotic is atleast minimally soluble, or of removing a nonvolatile solvent in whichthe antibiotic and lipid has been dissolved, typically by dialysis, havebeen eliminated. Instead, the present invention provides a new anduseful lipophilic charge complex of Amphotericin B which overcomes theprevious problems of insolubility. Additionally, the present inventionprovides a new and useful process for the production of Amphotericin Bliposomes suitable for scaling up production to commercial quantities.Additionally, the invention provides a method for production ofAmphotericin B liposomes of increased stability and decreased toxicity.

In one preferred embodiment of this invention, the new soluble complexis formed between Amphotericin B and distearoylphosphatidylglycerolwhich has been protonated during dissolution in a solution of chloroformand methanol acidified to a pH of about 1.0 to 3.0. The AmphotericinB--phospholipid complex, while in solution in the small amount ofacidified chloroform and methanol, can be mixed with phosphatidylcholineand cholesterol and reproducibly spray dried under controlled conditionsto yield a lipid powder which is readily processed into liposomes, usingan aqueous buffer solution having a pH such that the pH of the finalsolution is below about 5.5, preferably between about 4.5 and 5.5.Accordingly, the present invention allows commercial scaling up of newmaterials for liposomal production. Further, the liposomes formed withthis invention can be lyophilized and stored for later rehydration andinjection without significant change in size or toxicity. The advantagesof the present invention, will become clear after considering thefollowing detailed description.

DETAILED DESCRIPTION OF THE INVENTION

Distearoylphosphatidylglycerol, or other homologousphosphatidylglycerols, such as dilaurylphosphatidylglycerol,dimyristoylphosphatrdylglycerol or others, is dissolved in an equalvolume solution of chloroform and methanol and acidified before formingthe described soluble complex with Amphotericin B. Thus, 2.5 Nhydrochloric acid is added to the distearoylphosphatidylglycerol sodiumsalt solution in methanol:chloroform (1:1) to adjust the pH to between1.0 and 3.0, as measured on prewetted pH paper, prior to mixing withAmphotericin B, and thereby acidifying the phospholipid. TheAmphotericin B, or other polyene such as a tetraene, pentaene, orhexaene, is suspended in an equivolume solution of chloroform andmethanol and then added to the acidic distearoylphosphatidylglycerolsolution. Complex formation is facilitated by briefly warming thesolution to about 65° Centigrade. At this stage, the concentration ofAmphotericin B may be in excess of 45 mg/ml.

In the resulting solution additional lipids such as phosphatidylcholinesmay also be dissolved. Cholesterol or another sterol, such asergosterol, stigmosterol, or androsterone, is included to improve thestability of the resulting liposomes, and thus maintain the liposomeintact during circulation in the bloodstream. The solution is atranslucent orange. Typically, the Amphotericin B concentration in thefinal solution is greater than 25 mg/ml, and the total dissolved solidmaterial 15-20 percent by weight.

Among the additional phosphatidylcholines that may be dissolved in theAmphotericin B, acidic phosphatidylglycerol complex solution,hydrogenated egg phosphatidycholine, hydrogenated soya lecithin anddistearoyl or dipalmitoyl phosphatidylcholine are preferred suchmaterials. Hydrogenated natural phospholipids or saturated aliphaticphospholipids are believed to work well because the lack of double bondsin the side chains is thought to render the liposomes resistant tooxidation and more physically stable.

The organic solvents may be removed from the solution by rotaryevaporation in a round bottom flask leaving a dry film comprised of thecomplex and other lipid materials. Other equivalent methods of solventremoval are also suitable, such as drying under vacuum. Alternately, thesolution can be applied to a spray dryer and solvent removed in acontinuous process to produce large quantities of a free flowing yellowpowder for liposome preparation. This novel complex thus provides thelong awaited continuous production capability sought by industry.

After the last traces of organic solvent have been removed, the driedlipid complex powder may be stored for later use as a starting materialfor liposome preparation. This product affords the stability necessaryfor storage previously unavailable to the industry. Thus, the initialchemical steps need not be undertaken each time liposomes are desired.

Liposome preparation is accomplished by first hydrating appropriatequantities of the lipid complex powder with an aqueous buffer,preferably at about 65° C. Aqueous buffer solutions may also containsalts such as sodium chloride, or saccharides such as dextrose orlactose, to achieve any desired osmolarity. The pH of the solution iscarefully controlled to achieve a final solution having a pH of about5.5 or less, generally between about 2.0 and 5.5, preferably betweenabout 4.5 and 5.5

Liposomes are then formed by the application of shearing force.Typically shearing force can be applied using sonification orhomogenization, or by freezing and thawing, dialyzing away a detergentsolution from lipids, or other known methods used to prepare liposomes.The size of the liposomes, as well as whether they are multilamellar orunilamellar, can be controlled using a variety of known techniquesincluding the duration of sonication. See Gregoriadis, G. A. SimpleProcedure to Preparing Liposomes Capable of High EncapsulationEfficiency Under Mild Conditions. Liposome Technology, Gregoriadis, G.A. (ed.) CRC Press: Boca Raton, Fla. (1983). The present invention isadaptable, in particular, to scaling up production of the smallunilamellar liposomes disclosed in pending Vestar Research, Inc.application Ser. No. 899,064--Improved Treatment of Systemic FungalInfections with Phospholipid Particles Encapsulating Polyene AntifungalAntibiotics, incorporated here by reference. Such small liposomes can besterilized by filtration since their diameter is less than 0.2 μm.Virtually all of the initial Amphotericin B becomes associated with theliposome fraction when this technique is employed.

These liposome preparations, when formed in saccharide solutions such as9% lactose, may be lyophilized in vials under suitable conditions toform a dried yellow cake or plug of material. At a later time water maybe introduced into the vial to redissolve the solid cake and form asuspension of Amphotericin B liposomes suitable for injection.Lyophilization thus affords the clinician the significant advantage ofincreased convenience.

Amphotericin B, and other polyene antifungal antibiotics, areamphiphilic. One side of the macrocyclic compound is composed of aseries of unsubstituted hydrocarbons with double bonds while theopposing side is substituted with hydroxyl groups. Thus, the moleculestend to exhibit polarity, one side lipophilic and hydrophobic the otherside lipophobic and hydrophilic.

Furthermore, Amphotericin B has one carboxyl group, a Lowry-Br.oslashed.nsted acid, and one amine group, a Lowry-Br.o slashed.nstedbase. Therefore, in a neutral pH range of from 5 to 9 the carboxyl grouptends to give up a proton while the amine group tends to accept thatproton. The net result is that the molecule remains neutral anduncharged while at the same time having two ionized or charged groups--anegatively charged carboxyl group and a positively charged amine group.In that same neutral pH range phospholipid molecules, such asdistearoylphosphatidylglycerol are charged. They have an ionizedphosphate group giving the molecule a negative charge. Equally, suchphospholipid molecules are amphiphilic in that the long alphatic tailsare hydrophobic and lipophilic while the ionizable phosphate head is, ofcourse, hydrophilic and lipophobic.

When, however, the phospholipid molecule is solubilized in a proticsolvent with a pH between about 1.0 and 3.0, the phospholipid moleculetends to accept a proton and thus form a comparatively neutral molecule.When Amphotericin B is then added to the above acidified solution, theproton of the phosphate group will tend to be transferred to thecarboxyl group of the Amphotericin B. The result is that theAmphotericin B molecule will have a net positive charge. Concomitantly,the phospholipid's phosphate group will give up a proton and becomenegatively charged. The thus formed, oppositely charged, moleculesattract; their oppositely charged groups forming an ion pair.

The molecular attraction between Amphotericin B and phosphatidylglycerolis thus greatly increased. The aliphatic hydrocarbon chains of thephospholipids are attracted by hydrophobic interactions to the longchain of unsubstituted double bonds of the polyene. In the specificinstance of Amphotericin B, the molecule is a heptaene with seven doublebonded carbons along an unsubstituted section of 16 carbon atoms. In thespecific instance of distearoylphosphatidylglycerol there are 16unsubstituted methylene groups between the ester group and terminalmethyl group.

In addition to the hydrophobic interaction, the ionized groups form astrong association. In the protonating environment above described, theAmphotericin B will have a positive charge and the phosphatidylglycerolwill have a negative charge. Thus, the phosphatidylglycerol and theAmphotericin B will form a strong association.

The strongly associated complex so formed is highly soluble in smallamounts of organic solvent. Thus, the disadvantages of theaforementioned prior art methods have been overcome. See, for example,U.S. Pat. No. 4,663,167, May 5, 1987, Lopez-Berestein et al.Accordingly, the present invention provides a procedure for scaling-upproduction which the industry has long awaited.

A further aspect of the present invention is that the AmphotericinB--phosphatidylglycerol complex will associate with phosphatidylcholineand cholesterol during formulation and will not precipitate out of theorganic solvent solution. Furthermore, if the pH of this organic solventis maintained at 4.5 or less, the complex continues to remain stable andstrongly associated. During liposome hydration, the pH of the aqueousbuffer is controlled to give a solution having a final pH preferablybetween about 4.5 and 5.5. At that pH, the Amphotericin B-lipid complexis highly stable and has a high affinity for the lipid bilayer intowhich it becomes inserted. The result is a decrease in acute toxicity asdemonstrated by the following Chart 1:

    ______________________________________                                                    Liposome Preparation                                              Preparation Solution pH   LD50 (mg/kg)                                        ______________________________________                                        1           4.6           30                                                  2           4.8           >30*                                                3           5.1           >30*                                                4           5.6           20                                                  5           6.3           <10                                                 ______________________________________                                         Acute Toxicity Test on Mice using Liposomal Formulation Herein Described      while Varying Liposomal Preparation Solution pH.                              *The decrease in toxicity was so surprisingly great that no upper end         point was reached.                                                       

Thus, a significant decrease in toxicity is attained using the presentinvention. The decrease in toxicity allows for an increase in thetherapeutic dosages that may be safely administered and thus offersgreatly improved methods for Amphotericin B treatment. See U.S. Pat. No.4,663,167, May 5, 1987, Lopez-Berestein et al.

Furthermore, the associated complex is highly stable during storage. Theliposomes formed from either a film, or spray dried powder, afterhydration with a saccharide buffer, may be lyophilized. The lyophilizedcake can be stored preferably in a sterile lyophilization vial and laterrehydrated with sterile water for injection. The reconstituted liposomesretain therapeutic efficacy.

EXAMPLE 1 Formation of Amphotericin B--Phosphatidylglycerol Complex

632.7 mg distearoylphosphatidylglycerol sodium salt (Avanti PolarLipids, Birmingham, Ala.) was dissolved in 4 ml of an equivolumesolution of chloroform and methanol at 65° Centigrade. 300 ul 2.5 M HClwas added to the solution. 375.9 mg Amphotericin B (SquibbPharmaceuticals, New Brunswick, N.J.) was first suspended in 4.0 ml ofequivolume solution of chloroform and methanol, and then the suspensionwas added to the acidified DSPG solution. The Amphotericin B--DSPGlipophilic complex was formed with heating at 65° C. for several minutesyielding an orange solution of the dissolved Amphotericin B complex witha pH of approximately 1.5. The concentration of Amphotericin B wasapproximately 45 mg/ml.

1598.4 mg hydrogenated egg phosphatidylcholine (Avanti Polar Lipids) wasdissolved in 4.5 ml of an equivolume solution of chloroform and methanolat 65° C. to yield a clear solution. 393 mg cholesterol (Sigma ChemicalCo.) was also dissolved in a 4.5 ml equivolume solution of chloroformand methanol at 65° C. to yield a clear solution. The cholesterol andhydrogenated egg phosphatidylcholine solutions were then mixed with theAmphotericin B--DSPG complex solution giving a translucent orangesolution. 175 ul NaOH 2.5 M was then added to this solution to yield apH of approximately 4.5. The weight of total dissolved solids wasbetween 15 and 20% on a weight to volume basis.

The formulation in this example has the following molar ratio:

    ______________________________________                                        Amphotericin B         0.4                                                    Distearoylphosphatidylglycerol                                                                       0.8                                                    Hydrogenated Egg Phosphatidylcholine                                                                 2.0                                                    Cholesterol            1.0                                                    ______________________________________                                    

Other formulations are listed in Table 1.

Those formulations demonstrate that the invention is equally suitable toother formulations. The molar ratio of the primary component lipid tosterol may vary from at least 1:1 to 4:1. Similarly, the molar ratio ofpolyene to charged phospholipid may vary at least from 0.5:1 to 4:1.

Formation of Spray Dried Powder

The lipid solution containing the novel Amphotericin B--DSPG complexcontains only small amounts of organic solvent and consequently can bespray dried to a powder in a manner making the invention uniquelysuitable to continuous flow manufacturing procedures. The invention thusallows for scaled-up production of liposomes as compared to the priorart. The spray dried powder thus formed can be stored.

In one preferred embodiment, the lipid solution containing theAmphotericin B--DSPG complex was pumped as a fine mist into the spraydryer apparatus with an inlet temperature of 45° C. A free flowingyellow to light orange powder resulted. The powder so formed wascollected and stored at -20° C. in a desiccator.

Amphotericin B Liposome Preparation

The stored powder can then be hydrated in any quantity and used at anytime to form liposomes for treatment of fungal infection. In onepreferred embodiment, it is desirable to simultaneously sterilize thefinished liposome preparation. Thus, small unilamellar vesicles weredesired which could be sterilized by filtration through a 0.22 μm poresize filter.

15 gm of the spray dried powder was hydrated in 750 ml aqueous buffer of9% (w/v) lactose containing 10 millimolar sodium succinate at pH 5.5warmed to 65° C. for 40 to 60 minutes. The shearing force to form thesmall unilamellar vesicles was provided by a 10 minute exposure to ahigh shear force emulsification technique (see pending Vestar, Inc.application Ser. No. 696,727, now U.S. Pat. No. 4,753,788).

Characterization of Liposomes

The concentration of components of the above described preferredembodiment were determined by high performance liquid chromatography andare shown below. The mean liposome diameter was determined to be 38.3 nmby dynamic light scattering.

    ______________________________________                                        Component          Concentration                                              ______________________________________                                        Amphotericin B     1.86 mg/ml                                                 Hydrogenated Egg PC                                                                              10.12 mg/ml                                                Cholesterol        2.21 mg/ml                                                 Distearoyl Phosphatidyl glycerol                                                                 4.29 mg/ml                                                 ______________________________________                                    

EXAMPLE 2 Additional Formulation Compositions of AMB Liposomes

Several studies were performed in order to evaluate the effect ofaltering the ratios of various components in the liposome formulation.These studies provide evidence for the unique advantage the AmphotericinB phosphatidylglycerol complex provides in liposome preparation. Thus,preparation 1 in Table 1, in which phosphatidylglycerol was omitted, didnot form AmB liposome. However, when distearoyl phosphatidylglycerol wasadded in the molar ratio of 0.5 to 2.5 times that of Amphotericin B(preparations 2-6, Table 1) liposomes were formed.

The importance of the added cholesterol is illustrated in preparations7-10, Table 1. Although liposomes could be formed with the AmphotericinB-phosphatidylglycerol complex in the absence of cholesterol(preparation 7) or with a low concentration of cholesterol (preparation8), these preparations were more toxic than preparations 9 or 10 inwhich cholesterol content was increased. Thus, the molar ratio ofcholesterol to phosphatidylcholine was optimal in the range of 1:4 to1:1.

Alternatives to hydrogenated egg phosphatidylcholine were investigatedand results are summarized in Table 1, preparations 11 and 12.Hydrogenated soybean phosphatidylcholine and distearoylphosphatidylcholine formed satisfactory Amphotericin B liposomes.

Preparation 13 in Table 1 demonstrates that distearoylphosphatidylglycerol can be replaced by dilauroyl phosphatidylglycerol.The lipid soluble complex with Amphotericin B was formed andsatisfactorily incorporated into liposomes.

EXAMPLE 3 Antifungal Efficacy of Amphotericin B Liposomes

360.1 mg of the spray dried powder were hydrated at 65 degreesCentigrade for 40 minutes with 9% lactose containing 10 mM succinatebuffer, pH 5.62. Liposomes were prepared by sonication for four minuteswith a 1/2 probe at 65° C. under a nitrogen atmosphere. Three successivebatches of liposomes were prepared in a similar fashion. After sterilefiltration, the Amphotericin B concentration was determined to be 1.73mg/ml.

For therapeutic efficacy studies, groups of 8 mice were givenintravenous inoculations of 3.5×10⁵ Candida albicans yeast cells. Threedays post-infection, animals were treated with a single dose of eitherfree Amphotericin B or liposomal Amphotericin B. A severe systemicinfection existed in animals which were not treated until three dayspost infection. Each successive group was treated with an increasingdose of medicament in order to establish a dose response relationship.Twenty-nine days after infection the study was evaluated for survivinganimals. All untreated control animals had died by 8 dayspost-infection, with a median survival of 7 days. There was no doselevel of free Amphotericin B which produced any survivors at 29 dayspost infection. In contrast, all animals treated with 10 or 15 mg/kg ofliposomal Amphotericin B were still alive 42 days post infection. Thecomplete response to the free and liposomal Amphotericin B is shown inTable 2.

EXAMPLE 4 Stability of Liposome Amphotericin B to Lyophilization

The presence of lactose or other saccharides as excipients in theliposome Amphotericin B formulation serves to stabilize the integrity ofthe physical structure of the liposome during lyophilization. Thus, theformulations herein described can be lyophilized under appropriateconditions, and the lyophilized cake or plug can be reconstituted withsterile water at a later date.

The effect of lyophilization of four separate preparations of liposomeAmphotericin B has been evaluated. In all cases the preparationcontained 9% lactose as an excipient. In some cases, lyophilization andrehydration caused the mean liposome diameter to increase from about 40nm to about 70 nm. Acute toxicity increased from >30 mg/kg to 20-25mg/kg Amphotericin B when the rehydration was carried out at 22° C.However, if the same preparation was rehydrated at 65° C., there was notapparent reduction in toxicity (see experiment 3, Table 3).

Additional results are summarized in Table 3.

The foregoing description of the invention and the examplesdemonstrating the application of the invention to production ofAmphotericin B--lipid liposomes of illustrated size, structure andmedical utility are but exemplary of the various ways the invention canbe utilized. That other variations will be useful will be apparent tothose skilled in the art. Therefore, the present invention is to beconsidered limited only by the appended claims.

                                      TABLE 1                                     __________________________________________________________________________    PROPERTIES OF VARIOUS FORMULATIONS OF LIPOSOME AMPHOTERICIN B                                                       LIPOSOME                                                                            ACUTE                                                    MOLAR RATIO OF                                                                          % AMB                                                                              DIAMETER                                                                            TOXICITY                          PREPARATION                                                                           COMPONENTS     COMPONENTS                                                                              INCORP.                                                                            (NM)  (LD50)                            __________________________________________________________________________    1.      Hydr. Egg PC:Chol:DSPG:AMB                                                                   2:1:0:0.4 [DOES NOT FORM AMB LIPOSOMES]                2.      Hydr. Egg PC:Chol:DSPG:AMB                                                                   2:1:0.2:0.4                                                                             75   41    ND                                3.      Hydr. Egg PC:Chol:DSPG:AMB                                                                   2:1:0.4:0.4                                                                             91   41    ND                                4.      Hydr. Egg PC:Chol:DSPG:AMB                                                                   2:1:0.4:0.4                                                                             94   1.6   ND                                5.      Hydr. Egg PC:Chol:DSPG:AMB                                                                   2:1:0.8:0.4                                                                             100  39.3  ND                                6.      Hydr. Egg PC:Chol:DSPG:AMB                                                                   2:1:1:0.4 82   30.8  ND                                7.      Hydr. Egg PC:Chol:DSPG:AMB                                                                   2:0:0.8:0.4                                                                             82   53.6  <20 MG/KG                         8.      Hydr. Egg PC:Chol:DSPG:AMB                                                                   2:0.5:0.8:0.4                                                                           68   53.7  <20 MG/KG                         9.      Hydr. Egg PC:Chol:DSPG:AMB                                                                   2:1:0.8:0.4                                                                             85   40.3  >30 MG/KG                         10.     Hydr. Egg PC:Chol:DSPG:AMB                                                                   2:1.5:0.8:0.4                                                                           64   46.8  20 MG/KG                          11.     DSPC:Chol:DSPG:AMB                                                                           2:1:0.8:0.4                                                                             68   44.5  >30 MG/KG                         12.     Hydr. Soya PC:Chol:DSPG:AMB                                                                  2:1:0.8:0.4                                                                             100  42.9  >30 MG/KG                         13.     Hydr. Egg. PC:Chol:DLPG:AMB                                                                  2:1:0.8:0.4                                                                             84   41.4  ND                                __________________________________________________________________________     ND = Not Determined                                                      

                  TABLE 2                                                         ______________________________________                                        THERAPEUTIC EFFICIENCY OF LIPOSOMAL AMPHOTERICIN B                            IN C. albicans INFECTED MICE                                                              29 SURVIVORS.sup.1                                                            AMB                                                                           DOSE        3 DAY POST                                            TREATMENT   mg/kg       INFECTION THERAPY                                     ______________________________________                                        CONTROL.sup.2                                                                             --          0                                                     FREE AM B   0.5         0 (0%)                                                            1.0         0 (0%)                                                            1.5         0 (0%)                                                LIPOSOME    1.0         0 (0%)                                                AM B        2.5         3 (38%)                                                           5.0         4 (50%)                                                           7.5         6 (75%)                                                           10.0        8 (100%)                                                          15.0        8 (100%)                                                          20.0        4 (50%)                                               ______________________________________                                         .sup.1 n = 8 Animals per group.                                               .sup.2 Control median life span was 7 days.                              

                  TABLE 3                                                         ______________________________________                                        Effect of Lyophilization on Liposome Amphotericin B                                                           Acute                                                         Liposome Ampho B                                                                              Toxicity                                                                             Rehydration                            Ex-             Diameter Conc.  (LD50  Temperature                            periment                                                                             Conditions                                                                             (nm)     (mg/ml)                                                                              mice)  (degree C.)                            ______________________________________                                        1.     Before   38.3     1.86   >30 mg/kg                                            Lyo.                                                                          After Lyo.                                                                             69.9     1.60   >30 mg/kg                                                                            65                                     2.     Before   45.3     1.75   >30 mg/kg                                            Lyo.                                                                          After Lyo.                                                                             45.1     1.56   >30 mg/kg                                                                            65                                     3.     Before   42.4     1.72   >30 mg/kg                                            Lyo.                                                                          After Lyo.                                                                             66.9     1.72    25 mg/kg                                                                            22                                            After Lyo.                                                                             61.8     1.69   >30 mg/kg                                                                            65                                     4.     Before   40.3     1.88   >30 mg/kg                                            Lyo.                                                                          After Lyo.                                                                             70.6     1.90    20 mg/kg                                                                            22                                     ______________________________________                                    

We claim:
 1. A process for solubilizing a polyene antibiotic, comprisingforming a soluble complex between the polyene antibiotic and aphosphatidylglycerol in an acidified organic solvent and maintainingsaid soluble complex at a pH of 4.5 or less.
 2. The process of claim 1in which the soluble complex is formed in an organic solvent having a pHof from about 1.0 to about 3.0.
 3. The process of claim 1 or 2 in whichthe phosphatidylglycerol is selected from the group consisting ofdistearoylphosphatidylglycerol, dilaurylphosphatidylglycerol, anddimyristoylphosphatidylglycerol.
 4. The process of claim 1 or 2 in whichthe polyene antibiotic is amphotericin B.
 5. The process of claim 3 inwhich the polyene antibiotic is amphotericin B.
 6. The process of claim4 in which the phosphatidylglycerol is distearoylphosphatidylglycerol.7. A process for the formation of liposomes containing a polyeneantibiotic, comprising forming a soluble complex between a polyeneantibiotic and a phosphatidylglycerol in an acidified organic solventand maintaining said soluble complex at a pH of 4.5 or less, removingthe organic solvent to form a dried complex, and hydrating the driedcomplex with an aqueous buffer solution to form liposomes having adiameter of less than 2.0 μm.
 8. The process of claim 7 in which thecomplex is formed in an organic solvent having a pH of form 1.0 to 3.0.9. The process of claim 7 or 8 in which the phosphatidylglycerol isselected from the group consisting of distearoylphosphatidylglycerol,dilaurylphosphatidylglycerol, and dimyristoylphosphatidylglycerol. 10.The process of claim 7 or 8 in which a sterol is included with thesoluble complex to form liposomes containing a polyene antibiotic and asterol.
 11. The process of claim 9 in which a sterol is included withthe soluble complex to form liposomes containing a polyene antibioticand a sterol.
 12. The soluble complex of a polyene antibiotic and aphosphatidylglycerol, maintained in an acidified organic solvent havinga pH of 4.5 or less.
 13. The complex of claim 12 in which the polyeneantibiotic concentration is greater than 25 mg of antibiotic permilliliter of solvent.
 14. The process of claim 10 wherein said sterolis cholesterol.
 15. The process of claim 11 wherein said sterol ischolesterol.
 16. A method for the treatment of a disseminated fungalinfection comprising administering a therapeutically effective amount ofthe liposome prepared according to the process of claim 7.